Osteogenic protein-1 inhibits nucleus pulposus cell apoptosis through regulating the NF-κB/ROS pathway in an inflammation environment

Abstract

Background: Intervertebral disc degeneration is a pathological process that involves an inflammation response. As a classical cellular feature, several studies have demonstrated that inflammation can promote nucleus pulposus (NP) cell apoptosis. Therefore, attenuation of NP cell apoptosis may be a potential way to retard disc degeneration.

Objective: The present study was aimed to investigate the protective effects of osteogenic protein-1 (OP-1) against NP cell apoptosis in an inflammation environment, and the potential signaling transduction pathway.

Methods: Rat NP cells were cultured in medium with or without inflammatory cytokine tumor necrosis factor (TNF)-α for 6 days. The exogenous TNF-α was added into the medium to investigate its protective effects. NP cell apoptosis was evaluated by cell apoptosis ratio, caspase-3 activity, gene/protein expression of apoptosis-related molecules (Bcl-2, Bax, and caspase-3). Additionally, the intracellular reactive oxygen species (ROS) content and activity of the NF-κB pathway were also analyzed.

Results: Compared with the control NP cells, TNF-α significantly increased cell apoptosis ratio, caspase-3 activity, gene/protein expression of Bcl-2, Bax and caspase-3, ROS content, and activity of the NF-κB pathway. However, OP-1 partly attenuated these effects in NP cells treated with TNF-α.

Conclusion: OP-1 is effective in attenuating TNF-α-caused NP cell apoptosis, and the ROS/NF-κB pathway may be the potential signaling transduction pathway. The present study indicates that OP-1 may be helpful to inhibit inflammation-mediated disc degeneration.

Keywords: TNF-α; apoptosis; disc degeneration; nucleus pulposus; osteogenic protein-1.

Figure 1. Osteogenic protein-1 (OP-1) decreased apoptosis ratio of TNF-α-treated nucleus pulposus (NP) cells

NP cell apoptosis was measured by flow cytometry. Data are shown as mean ± SD, n=3. *: Indicates a significant difference (P<0.05).

Figure 2. Osteogenic protein-1 (OP-1) decreased caspase-3 activity of TNF-α-treated nucleus pulposus (NP) cells

Caspase-3 activity was measured using a chemical kit. Data are shown as mean ± SD, n=3. *: Indicates a significant difference (P<0.05).

Figure 3. Osteogenic protein-1 (OP-1) decreased intracellular reactive oxygen species (ROS) content of TNF-α-treated nucleus pulposus (NP) cells

ROS content was analyzed using a chemical kit. Data are shown as mean ± SD, n=3. *: Indicates a significant difference (P<0.05).

Figure 4. Osteogenic protein-1 (OP-1) exhibited an anti-apoptotic gene expression profile in TNF-α-treated nucleus pulposus (NP) cells

Gene expression of apoptosis-related molecules (Bcl-2, Bax, and caspase-3) in NP cells was analyzed by real-time PCR. Data are shown as mean ± SD, n=3. *: Indicates a significant difference (P<0.05).

Figure 5. Osteogenic protein-1 (OP-1) exhibited an anti-apoptotic protein expression profile in TNF-α-treated nucleus pulposus (NP) cells

Protein expression of apoptosis-related molecules (Bcl-2 and Bax) in NP cells was analyzed by Western blot. Data are shown as mean ± SD, n=3. *: Indicates a significant difference (P<0.05).

Figure 6. Osteogenic protein-1 (OP-1) decreased activity of the NF-κB pathway in TNF-α-treated nucleus pulposus (NP) cells

Activity of the NF-κB pathway was expressed as the ratio of p-NF-κB protein expression to NF-κB protein expression. Data are shown as mean ± SD, n=3. *: Indicates a significant difference (P<0.05).